大黄灵仙方调控肝内胆管细胞炎症大鼠的作用及机制研究

Study on the effect and mechanism of Dahuang Lingxian formula on regulating intrahepatic bile duct cell inflammation in rats

目的:观察大黄灵仙方对肝内胆管细胞炎症模型大鼠MAPK/NF⁃κB信号通路中IKKα、ASK1、MKK3以及CX3CL1关键因子的表达水平的影响,探讨其缓解肝内胆管细胞炎症的改善作用及可能机制。方法:45只SD大鼠按照完全随机方法分为9组,每组大鼠5只,空白组、模型组、大黄灵仙颗粒组、NF⁃κB组、p38MAPK组、NF⁃κB+p38MAPK组、NF⁃κB+大黄灵仙颗粒组、p38MAPK+大黄灵仙颗粒组、NF⁃κB+p38MAPK+大黄灵仙颗粒组。除空白组外,余下各组大鼠均在胆总管注射5 mg/kg LPS制备肝内胆管炎症模型。第7天灌胃结束后取大鼠胆管树,采用HE染色观察其炎性程度,蛋白免疫印迹法和实时荧光定量PCR检测IKKα、ASK1、MKK3以及CX3CL1蛋白及mRNA表达量。结果:HE病理结果显示,模型组的肝内胆管组织和细胞炎症明显,加入大黄灵仙颗粒和信号阻断剂后肝内胆管炎症状态较前改善,总病理评分差异有统计学意义(P<0.05)。与模型组比较,大黄灵仙颗粒组、p38MAPK组、NF⁃κB组、NF⁃κB+p38MAPK组、NF⁃κB+大黄灵仙颗粒组、p38MAPK+大黄灵仙颗粒组、NF⁃κB+p38MAPK+大黄灵仙颗粒组的ASK1、CX3CL1蛋白及mRNA表达量下降,MKK3 mRNA表达量上升以及p38MAPK组IKKα和NF⁃κB+大黄灵仙颗粒组MKK3蛋白表达量上升(P<0.05)。与大黄灵仙颗粒组比较,p38MAPK组、NF⁃κB组、NF⁃κB+p38MAPK组、p38MAPK+大黄灵仙颗粒组、NF⁃κB+大黄灵仙颗粒组、NF⁃κB+p38MAPK+大黄灵仙颗粒组的ASK1、CX3CL1 mRNA表达量下降,p38MAPK+大黄灵仙颗粒组、NF⁃κB+大黄灵仙颗粒组、NF⁃κB+p38MAPK+大黄灵仙颗粒组的MKK3 mRNA表达量上升(P<0.05)。与大黄灵仙颗粒组相比,p38MAPK组的IKKα蛋白表达量下降,NF⁃κB+大黄灵仙颗粒组的ASK1、MKK3蛋白表达量上升(P<0.05),而NF⁃κB+p38MAPK+大黄灵仙颗粒组的CX3CL1蛋白表达量下降(P>0.05)。结论:大黄灵仙方可缓解LPS诱导的大鼠肝内胆管炎症反应,促进胆管细胞的修复,从而达到降低PIS发生及术后复发的目的,其作用机制可能与抑制NF⁃κB/MAPK信号通路中的IKKα、ASK1、MKK3以及CX3CL1关键炎症因子的活化有关。

Objective:To observe the effects of Dahuang Lingxian formula on the expression levels of IKKα,ASK1,MKK3 and CX3CL1 key factors in the MAPK/NF⁃κB signaling pathway in the intrahepatic cholangiocyte inflammation model of rats,and to explore the ameliorative effects and possible mechanisms of alleviating intrahepatic cholangiocyte inflammation.Methods:45 SD rats were divided into nine groups according to the complete randomization method,with 5 rats in each group:the blank group,the model group,the Dahuang Lingxian Granules group,the NF⁃κB group,the p38MAPK group,the NF⁃κB+p38MAPK group,the NF⁃κB+Dahuang Lingxian Granules group,the p38MAPK+Dahuang Lingxian Granules group,the NF⁃κB+p38MAPK+Dahuang Lingxian Granules group.Except for the blank group,rats in the remaining groups were injected with 5 mg/kg LPS into the common bile duct to prepare an intrahepatic bile duct inflammation model.After the intragastric admin⁃istration on the 7th day,the rat bile duct tree was removed,and HE staining was used to observe the degree of inflammation.Western blotting and real⁃time fluorescence quantitative PCR were used to detect the protein and mRNA expression of IKKα,ASK1,MKK3,and CX3CL1.Results:HE pathology results showed that the inflammation of intrahepatic bile duct tissue and cells in the model group was obvious.After adding Dahuang Lingxian Granules and signal blockers,the inflammation status of in⁃trahepatic bile ducts was improved compared with before,and the difference in total pathological scores was statistically significant(P<0.05).Compared with the model group,Dahuang Lingxian granules group,p38MAPK group,NF⁃κB group,NF⁃κB+p38MAPK group,NF⁃κB+Dahuang Lingxiang ranules group,p38MAPK+Dahuang Lingxian granules group,NF⁃κB+p38MAPK+Dahuang Ling The protein and mRNA expression of ASK1 and CX3CL1 in the Xian Granule group decreased,the expression of MKK3 mRNA increased,and the expression of IKKαin the p38MAPK group and NF⁃κB+MKK3 protein in the Da⁃huang Lingxian Granule group increased(P<0.05).Compared with the Dahuang Lingxian Granules group,The expression of ASK1 and CX3CL1 mRNA decreased in the p38MAPK group,the NF⁃κB group,the NF⁃κB+p38MAPK group,the p38MAPK+Dahuang Lingxian Granules group,the NF⁃κB+Dahuang Lingxian Granules group,the NF⁃κB+p38MAPK+Da⁃huang Lingxian Granules group.The expression of MKK3 mRNA in the p38MAPK+Dahuang Lingxian Granules group,NF⁃κB+Dahuang Lingxian Granules group,and NF⁃κB+p38MAPK+Dahuang Lingxian Granules group increased(P<0.05).Compared with the Dahuang Lingxian Granules group,the IKKαprotein expression in the p38MAPK group decreased,and the ASK1 and MKK3 protein expressions in the NF⁃κB+Dahuang Lingxian Granules group increased(P<0.05),while the NF⁃κB+p38MAPK+Dahuang Lingxian Granules group increased.The expression of CX3CL1 protein in the granule group decreased(P>0.05).Conclusion:Dahuang Lingxian Recipe can alleviate the LPS⁃induced intrahepatic bile duct inflammatory reaction in rats and promote the repair of bile duct cells,thereby achieving the purpose of reducing the occurrence and postoperative recurrence of PIS.Its mechanism of action may be related to inhibiting the NF⁃κB/MAPK signaling pathway.It is related to the activation of key in⁃flammatory factors such as IKKα,ASK1,MKK3 and CX3CL1.